In most living organisms, genetic information is stored in the form of DNA. This DNA is transcribed into messenger RNA (mRNA) which then is translated into protein. In eukaryocytes, there is usually some loss of genetic information in the process of converting genomic DNA to the mature mRNA. This loss can be due to introns/exons, RNA splicing, or protein processing. Therefore, the genetic basis of protein structure is more advantageously studied using mRNA than genomic DNA.
Unfortunately, mRNA is extremely unstable and easily digested by various ribonuclease (RNA digesting) enzymes (hereafter referred to collectively as "RNase") making experimentation difficult. Thus, many researchers have chosen to study DNA copies (cDNA) of interesting mRNA molecules. These copies are made using the enzyme reverse transcriptase (RT), isolated from retrovirus', which produces a single stranded DNA copy of the target mRNA.
Double stranded DNA (ds-cDNA) is generally more stable than single stranded cDNA (hereafter called "ss-cDNA"). The method of converting ss-DNA to ds-DNA using the enzyme DNA polymerase is well known in the art. The genetic information stored on ds-cDNA can then be used in various protocols, for example inserting ds-cDNA into expression vectors, which are then introduced into various cells. Promoters and restriction sites located on these vectors can then be used as tools for studying transcription and translation of the ds-cDNA.
In addition to using vector based promoters for transcription of mRNA nucleotides containing the polypeptide coding sequence (sense mRNA), antisense mRNA transcribed from the complementary cDNA strand can also be produced. Antisense mRNA is a useful tool for understanding biological function of a protein or mRNA whose function is unknown. Antisense RNA molecules are used to block production of specific proteins by anneling to the target mRNA and inhibiting translation. Therefore, it is anticipated that this type of translational inhibition will be important in the study of various gene products associated with a variety of diseases states.
Besides being a used for DNA polymerase based catalysis of ds-cDNA synthesis, ss-cDNA can be used as a template for polymerase chain reaction (hereafter called "PCR"). This method allows the rapid reproduction and enhancement of a specific gene sequence through the use of opposing nucleotide primers and a heat stable DNA polymerase. Multiple cycles of annealing and DNA synthesis rapidly produces many copies of the targeted gene. Thus, sense ss-cDNA can be used to specifically amplify a segment of its corresponding ds-cDNA.
One currently known protocol for producing ds-cDNA is the liquid phase method described in Sambrook, et al., "Molecular Cloning, A Laboratory Manual, 2d Ed.," Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) (hereinafter referred to as "Molecular Cloning"). The complete disclosure of this manual is incorporated herein by reference thereto. In the liquid phase method, antisense ss-cDNA is produced from mRNA using reverse transcriptase, followed by mRNA digestion with RNase. Ds-cDNA is then synthesized from complementary chains of the remaining antisense ss-cDNA using DNA polymerase. DNA polymerase reactions following reverse transcriptase don't require a specific primer since the RT leaves synthsized ss-cDNA with a self-priming loop structure at the 3' end.
With the liquid phase method, the resulting ds-cDNA does not contain a marker to identify the molecule's orientation. Accordingly, the newly synthesized ds-cDNA cannot be readily inserted into a cloning vector since 50% of the clones will be in the wrong orientation for transcription. A rapid method of directionally cloning mRNA into a vector would therefore be desirable.
Ss-cDNA or ds-cDNA can also be produced using solid phase material. (I. Raineri et al. in Nucleic Acids Research, 19:4010 (1991)). In the solid phase method, polydeoxythymidylic acid (poly dT), usually immobilized onto porous beads, is hybridized to the polyadenlic acid (poly A) tail of mRNA. Then, antisense ss-cDNA is synthesized from the bound polyadenelated mRNA by reverse transcriptase. After the template RNA is digested, second strand cDNA is synthesized by DNA polymerase. The resultant ds-cDNA has its antisense strand immobilized to the beads.
The solid phase method, however, yields product that cannot be disassociated from the insoluble support. In order circumvent this problem, the ss-cDNA can be released from the support-immobilized ds-cDNA by heating, and thereafter used to synthesize ds-cDNA using PCR. However, this adds another step to the reaction and requires an appropriate set of primers for every PCR reaction.
There is therefore a need for a simple method of creating unbound ds-cDNA clones from isolated mRNA. This method should preferably result in unbound, directionally cloneable products.
There are also various methods known of synthesizing mRNA from cDNA clones, one example is the liquid phase method which was described by S. Shichijo, et al. in Journal of Neuroscience Research, 30:316-320 (1991). In this method, ds-cDNA is inserted into a vector containing a RNA promoter. The vector is then linearized by restriction enzyme digestion and mRNA is synthesized using RNA polymerase. The synthesized mRNA is then treated with a deoxyribonuclease (DNA digesting) enzyme (hereafter called "DNase") to remove the template DNA. If necessary, a polyadenelated tail can be added to the end of the newly synthesized RNA using the enzyme Terminal Transferase and dATP's.
The solid phase method of producing mRNA is also well known. Hironori Terada, et al., "Movement analysis of transcription by immobilized DNA," Biophysics 31:49-52 (1991) describe one such method. In this method, a DNA sequence is digested from the genome of bacteriophage .lambda. by restriction enzyme cleavage, resulting in random DNA fragments with "sticky ends. T4 DNA polymerase is then used to fill in these sticky ends using biotinylated dUTP. The restriction enzyme is selected so as to leave a sticky end with an exposed dA nucleotide to which the biotinylated dUTP will hybridize during synthesis of DNA by the T4 DNA polymerase. Random sequences are then immobilized to an acrylamide support bearing avidin. mRNA can then be synthesized from those sequences bearing a natural .lambda. promoter sequence using a polymerase T7 RNA polymerase or SP6 RNA polymerase. This system was designed to study the kinetic analysis of transcription in bacteriophage .lambda..
Both the liquid phase and solid phase methods of mRNA synthesis have disadvantages. The liquid phase method requires the use of a vector containing a RNA promoter, and also requires that the vector be converted into a linear sequence after insertion. The solid phase method does not always provide complete genetic information because the source of this information is immobilized genomic DNA and not mRNA. Thus, improved methods of mRNA synthesis would be desirable.
Stamm et al. in Nucleic Acids Research, 19:1350 (1991) described oligonucleotides covalently coupled via an amino link to a solid support for use in facilitating PCR experiments. However, the technique described by Stamm et al. does not specifically provide for the production of cDNA or RNA. Moreover, no mechanism is provided for removing the oligonucleotides covalenty coupled to the support.